Abstract
Background:
The parasitic disease Theileria ovis (Ovine Theileriosis) affects the health and productivity of infected sheep and sheep-based industries, especially in areas impacted by endemic tick-borne disease. Despite the availability of this parasite and its significant economic and veterinary impact, some molecular data, especially on T. ovis, genetic diversity, and phylogeny in sheep in Iraq, are scarce. Definite molecular description is critical to understanding the parasite’s biology, especially in terms of its transmission and genetic diversity at the regional and local levels.

Aim:
This study aimed to achieve molecular characterization of T. ovis in sheep in Central Iraq and analyze the genetic diversity and phylogenetics of the local isolates through partial 18S ribosomal RNA (rRNA) gene sequencing.

Methods:
A complete blood sample was collected from the sheep, from which genomic DNA was extracted. For the molecular characterization of T. ovis, a nested conventional PCR was performed targeting the 18S rRNA gene. Positive PCR amplicons were selected and subjected to Sanger sequencing, after which the sequencing data were edited and compared to GenBank and other countries’ data using NCBI-BLAST. The phylogenetic analysis was based on the Maximum Likelihood method and the Tamura -Nei model. Sequence alignment was performed to determine the variation and similarity in nucleotides among the different isolates.

Results:
The expected 520-bp fragment of the 18S rRNA gene in the sheep samples was successfully amplified using nested PCR, confirming the presence of T. ovis infections. Ten representative isolates were sequenced, and the resulting sequences were submitted to GenBank with the following accession numbers: PX755062 to PX755071. A BLAST analysis of the submitted sequences showed a high nucleotide identity percentage to the previously characterized T. ovis isolates from different countries, ranging from 98.81% to 100%. The isolates from Egypt and Türkiye exhibited the greatest similarity, whereas the isolates from Pakistan, China, Japan, and Iraq had slightly lower identity values than the isolates from other countries. Phylogenetic analysis indicated that the T. ovis clade also contained the Iraqi sheep isolates, and in conjunction with the global reference strains, the identity of the T. ovis species was confirmed, indicating a low level of genetic diversity. The presence of a great deal of sequence conservation across the sheep isolates was demonstrated by the multiple sequence alignment, which was the result of minor nucleotide substitutions that did not change the species-level classification.

Conclusion:
This study offers clear molecular data regarding the circulation of T. ovis in sheep in central Iraq. Iraqi isolates and global strains have high genetic similarity. This proves that a conserved 18S rRNA gene with minor regional variation exists. The data hydra provides to the epidemiology of ovine theileriosis in Iraq is significant and exemplifies the need for complex molecular surveillance to monitor and control the disease.

Key words: Theileria ovis; Sheep; Nested PCR; 18S rRNA gene; Genetic diversity; Phylogenetic analysis; Iraq.